KR20230109549A - Novel biocompatible composition for labelling a lesion and macufacturing method thereof - Google Patents

Abstract

The present invention is a microsphere particle containing a biocompatible polymer and a pigment for staining living tissue; And a surface modifier coated on the surface of the microsphere particles; a composition for marking a target lesion and a method for preparing the same, which are directly administered to the target lesion during surgery to determine the size and location of the target lesion in real time it's about The composition for marking the target lesion of the present invention, when injected into the target lesion, stays continuously for up to several months and can detect the location and size of the target lesion in real time during surgery, thereby improving the success rate of surgical operation of the target lesion as well as However, since it can prevent excessive loss of normal tissue, it can be widely used as an effective surgical marker.

Description

Composition for labeling biocompatible target lesions and method for producing the same

The present invention relates to a composition for labeling a bio-friendly target lesion and a method for manufacturing the same, and more particularly, to a composition for labeling a target lesion in which a pigment for staining a living tissue and a bioprotein are included in a bio-friendly polymer, and using the composition for labeling the target lesion The present invention relates to a method of imaging labeled information on a lesion site and a marker for surgery of a target lesion in which the composition for marking the target lesion and a biopolymer are mixed to improve in vivo stability and imaging capability.

In the case of chemotherapy, methods using various anticancer agents have been developed, but a method of removing cancer cells through a surgical method is still most commonly used. However, when using a surgical method, a technique for minimizing the surgical range during surgery is essential for the health and welfare of the patient after surgery. In particular, in the case of breast cancer, the goal of breast-conserving treatment can be achieved only when the number of lesions to be resected during surgery for small breasts in Korea is smaller than in other countries. The surgical scope is determined by the lesion and the extra marginal area around the lesion. If the surgeon does not know exactly the area and extent of the lesion, he or she has to leave a large margin around the lesion. This is because if the surgical scope is recklessly reduced, the tumor may remain on the resection margin. However, in actual clinical surgery, there are not many methods for the operating surgeon to accurately check the lesion in real time during surgery. Although very precise diagnostic methods have been developed, these diagnostic methods cannot be used during surgery, so during actual surgery, the surgeon mainly relies on the operator's sense of touch or vision. In particular, when the lesion is small, it is more difficult to differentiate. In order to achieve the purpose of micro-invasive surgery and surgery, a technique for informing an operator of a lesion during surgery is required.

In conventional tumor removal surgery, especially breast cancer surgery, the location of the patient's microlesion is confirmed by ultrasound, mammography, or magnetic resonance imaging before surgery, the location of the identified lesion is marked, and then tissue in the marked area is removed. However, as a method of marking the position of the confirmed lesion, a method of drawing a picture on the skin surface, a method of using a wire, a method of injecting a black pigment such as charcoal, etc. are used. However, the method of drawing a picture using a pen on the skin to mark the location of the lesion, due to the nature of very flexible breast tissue, the shape of the breast changes greatly during diagnosis and in the operating room. Due to the fact that only marking on the skin surface is insufficient, it can be used most easily, but has the disadvantage of the lowest accuracy. Next, the method of piercing the breast lesion using a wire originally requires the wire to be inserted vertically into the skin surface, but it is inevitably inserted obliquely because it can affect the ultrasound probe, and the location of the wire determines the movement of the breast. There are disadvantages in that it shows lower accuracy than expected due to the fact that it can move according to, etc., the inserted wire interferes with surgery, and a procedure to additionally resect the insertion site of the wire must be performed. Finally, the method of injecting a pigment such as charcoal has the advantage that the injected pigment binds to the lesion and accurately marks the location of the lesion. There is a disadvantage that the surgical site may be contaminated by the pigment. The above-listed disadvantages can be commonly applied even during a surgical operation for removing cancer tissues other than breast cancer.

As such, it is difficult to precisely determine the surgical range for surgically removing cancer lesions with the technology developed so far. However, after surgery, an examination to confirm whether the lesion was normally removed should be followed.

Under this background, the present inventors have developed a marker (KR-10-2012-0153793), which uses a complex in which a pigment for staining living tissue is combined with albumin of the corresponding group as a marker when the cancer lesion is surgically removed. It was confirmed that the lesion could be effectively adsorbed to accurately mark the location of the lesion, and the range of the lesion to be removed could be accurately identified by tracking the pigment in real time during surgery.

In general, dyes or fluorescent dyes do not stay long at the injection site, spread to the surroundings within a few hours, and are eventually excreted into the body, losing the ability to mark lesions. In the case of the existing invention (KR-10-2012-0153793), compared to the existing general new (fluorescent) pigment, it stays on the injection site for a long time, but it is difficult to exceed several days. This is especially true when the injection site is injected into a tissue that is constantly moving or has a lot of blood flow, such as the stomach, or where the tissue is relatively loose and not rigid. That is, conventional markers have a problem in that the ability to label lesions is lost in a short period of time as the pigment does not stay at the injection site for a long time and diffuses to the surroundings within several hours.

Among cancer patients, when the lesion is so large that surgery cannot be performed immediately, chemotherapy is performed first and then surgery to remove the cancer is increasing. At this time, the lesion is marked in advance before chemotherapy, which takes several months, and the area is excised after chemotherapy. At this time, if the tumor is well treated with chemotherapy and is reduced to a fine size, it is often difficult to find the cancer site during surgery. A metal clip is placed during chemotherapy to check the cancer site, but it is not easy to find a metal clip in actual surgery, and it is not uncommon for it to move during a long period of chemotherapy, so improvement measures are needed. It is very useful if the fluorescent dye can stay in the injected area for several months and the fluorescence or color can be confirmed several months later.

In order for the (fluorescent) pigment to stay at the injection site for a long time, it must be particles of an appropriate size or bonded to such particles (diameter: several to hundreds of micrometers) so that they do not spread to the surroundings, and the pigment is decomposed or denatured by biological reactions to be stable for a long period of time, or fluorescence should not be lost.

Microspheres of general micrometer-sized particles have been developed in various materials. Various porous microspheres have been developed as microspheres capable of carrying (fluorescent) dyes, but these are mainly used to release the supported material, here the (fluorescent) dyes, at a slow and controlled rate. It is not mainly for the purpose of not releasing the drug, but for the purpose of properly releasing it, which is completely different from the required purpose. That is, the (fluorescent) pigment must be in a form in which the microspheres are not discharged into the living body, and the microspheres must be made of a material usable in the living body.

Under this background, the easiest way to devise is to make microspheres by mixing (fluorescent) pigments with biocompatible polymers. However, the fluorescence is lost, so the purpose cannot be achieved.

The present invention has devised a method of completely embedding and encapsulating a (fluorescent) pigment by dissolving a biocompatible polymer in a solvent and utilizing a double emulsion method. can solve

In addition, a method for stabilizing the encapsulated (fluorescent) dye in the polymer microspheres for a long period of time was devised, and it was confirmed that color or fluorescence can be displayed for a longer period of time if the dye is combined with a protein rather than the dye alone.

In addition, if the surface is modified with hyaluronic acid, after injection, fibroblasts are activated in the surrounding tissue to generate collagen tissue, which can be well adhered. However, since hyaluronic acid is insoluble in organic solvents, it is difficult to coat the polymeric microspheres with hyaluronic acid using a general method. A method of dissolving hyaluronic acid or collagen in a water-soluble solvent in the process of manufacturing microspheres was devised, and a manufacturing method that can be coated with hyaluronic acid or collagen in the process of making biocompatible polymer microspheres was developed.

Patent Document 1: KR 10-2012-0153793

The present invention has been derived to solve the above problems, and an object of the present invention is to provide a composition for labeling lesions in the body comprising a biocompatible polymeric synthetic material including a pigment or fluorescent pigment and a bioprotein.

Specifically, the object of the present invention is (a) to provide a method for keeping lesions in the body at the injection site for several months or longer with color or fluorescent dye, (b) a method for preparing the compound for marking the lesion in the body and composition of the injection ( c) It is to provide a method of providing information on the location of the lesion using this.

As a means for achieving the above object, the present invention is a microsphere particle containing a biocompatible polymer and a dye for staining living tissue; and a surface modifier coated on the surface of the microsphere particles, and is directly administered to the target lesion during surgery for the target region to confirm the size and location of the target lesion in real time.

Here, the biocompatible polymer is polymethyl methacrylate (PMMA), polyglycolic acid (PGA), polylactic acid (PLA), polylactic acid-polyglycolic acid copolymer (polylactic acid- polyglycolic acid copolymer (PLGA), poly-ε-caprolactone (PCL), lactic acid-ε-caprolactone copolymer (PLCL), polydioxanone (PDO) ), polytrimethylene carbonate (PTMC), polyamino acid, polyanhydride, polyorthoester, polyphosphazene, polyiminocarbonate, It may be any one of polyphosphoester, polyhydroxyvalate, copolymers thereof, and mixtures thereof.

Here, the dye for dyeing the living tissue may be a dye or a fluorescent dye that can be confirmed with the naked eye.

Here, the pigments that can be confirmed with the naked eye are neutral, Nile blue, Bismarck brown, lithium carmine, trypan blue, Janus green, methyl violet, o-lamin, maragaite green, safranin, eosin, congo red, erythrosin, and nigrosin. , alcian blue hematoxylin, aniline blue, light green, and any one selected from the group consisting of combinations thereof.

Here, the fluorescent dye may be a near-infrared fluorescent dye.

Here, the fluorescent dye may be indocyanine green (ICG).

Here, the fluorescent dye may be characterized in that it is used alone or conjugated with a biological protein.

Here, the surface modifier may be any one of hyaluronic acid, collagen, and mixtures thereof.

Here, the microsphere particle may further include a biological protein.

Here, the biological protein may be albumin.

Here, the microsphere particles may have a diameter of 10 to 100 μm.

Here, the composition may have an average density of 1 to 3 g/mL.

In addition, the present invention is a means for achieving the above object, mixing an organic solvent to which a biocompatible polymer is added and distilled water to which a dye for dyeing living tissue is added; and injecting the mixed solution into an aqueous solvent to which a surface modifier is added, followed by stirring to form the microsphere particles, characterized in that the surface modifier is coated on the surface of the microsphere particles, for target lesion marking. A method for preparing the composition is provided.

Here, the preparation method of the target lesion-labeling composition may further include selecting microsphere particles having a specific size by washing and filtering the stirring solution.

Here, the distilled water may be further added with a pigment-biological protein conjugate for staining living tissue.

Here, the organic solvent is any one selected from the group consisting of dichloromethane (DCM), orthoxylene (o-xylene), m-xylene (m-xylene), para-xylene (p-xylene), and combinations thereof. can

Here, the concentration of the dye for staining living tissue may be 1 to 100 μM.

Here, the water-soluble solvent is polyvinylpyrrolidonem (PVP), polyacrylic acid (PAA), polyacryl amide (PAAm), polyhydroxyethyl methacrylate (PHEMA), poly It may be any one selected from the group consisting of polyvinyl alcohol (PVA) and combinations thereof.

Here, the concentration of the water-soluble solvent may be 0.1 to 10% w/w, and the concentration of the surface modifier may be 0.01 to 0.3% w/v.

In addition, the present invention is a means for achieving the above object, the composition for labeling the target lesion of claim 1; and hyaluronic acid (HA) having an average molecular weight of 0.5 to 3.0 MDa.

Here, based on 100% by weight of the injectable composition, hyaluronic acid (HA) may be included in an amount of 0.3 to 1% by weight.

Here, the hyaluronic acid (HA) may have a viscosity of 24 to 1500 cps at room temperature.

Here, the target lesion to which the injectable composition is injected may maintain a label for 4 to 12 months.

In addition, the present invention comprises the steps of directly administering the composition for marking the target lesion according to the present invention to the target lesion; And recognizing the position where the color changes from the tissue to which the composition for marking the target lesion is administered as the target lesion, and confirming in real time during operation of the marked area; a method for providing information on the location of the target lesion, including to provide.

The composition for marking the target lesion of the present invention, when injected into the target lesion, stays continuously for up to several months and can detect the location and size of the target lesion in real time during surgery, thereby improving the success rate of surgical operation of the target lesion as well as However, since it can prevent excessive loss of normal tissue, it can be widely used as an effective surgical marker.

1 is a schematic diagram showing a method for preparing a composition for labeling lesions for biocompatible purposes according to the present invention.
Figure 2 is a schematic diagram showing in detail the manufacturing method of the composition for labeling biocompatible lesions of the present invention.
Figure 3 (a) is a real picture of PMMA including ICG and HSA and a picture taken with a fluorescence camera.
Figure 3 (b) is a photograph taken with a fluorescence microscope of PMMA according to the presence or absence of ICG and HSA.
Figure 4 is a graph showing the density according to the microsphere particle concentration of the present invention for distilled water.
5 is a graph showing the change in near-infrared fluorescence signal intensity of microspheres according to the concentration change of ICG and albumin.
6 is a photograph showing the degree of dispersion of microspheres according to the concentration of hyaluronic acid in the injectable composition for labeling biocompatible lesions of the present invention.
7 is a graph showing a comparison of changes in the intensity of near-infrared fluorescence signals of ICG, ICG-HSA, and ICG-HSA-PMMA particles over time under in vitro conditions measured by IVIS (Perkin Elmer, Hopkinton, MA).
Figure 8a is a photograph showing the comparison of particle stability over time after putting ICG-HSA-PMMA particles in HSA and distilled water, respectively.
Figure 8b is a photograph showing the stability of ICG-HSA-PMMA particles with the passage of time after putting them in an agarose gel.
9 is a result of IVIS measurement and comparison of the intensity change of the near-infrared fluorescence signal over time when ICG-HSA-PMMA particles injected into ICR mice were injected with hyaluronic acid or distilled water in vivo.
10 shows the change in the intensity of the near-infrared fluorescence signal over time when the ICG-HSA-PMMA particles injected into the ICR mouse were injected with hyaluronic acid or distilled water under in vivo conditions, photographed with a fluorescence camera and analyzed with the Image J program. This is the result of taking a graph and a mouse with a fluorescence camera.

Since the present invention can make various changes and have various embodiments, specific embodiments will be illustrated in the drawings and described in detail in the detailed description. However, this is not intended to limit the present invention to specific embodiments, and should be understood to include all modifications, equivalents, or substitutes included in the spirit and technical scope of the present invention.

Throughout the present specification, when a certain component is said to "include", it means that it may further include other components without excluding other components unless otherwise stated.

As used herein, the terms "about," "substantially," and the like are used at or approximating that number when manufacturing and material tolerances inherent in the stated meaning are given, and are intended to assist in the understanding of this disclosure. Accurate or absolute figures are used to prevent undue exploitation by unscrupulous infringers of the stated disclosure. Also, throughout the present specification, "step of" or "step of" does not mean "step for".

Since those skilled in the art can make various applications through the gist of the present invention, the scope of the present invention is not limited to the following examples. The scope of the rights of the present invention extends to the obvious part that a person having ordinary knowledge belonging to the technical field of the present invention can easily substitute or change using the prior art based on the matters described in the claims.

Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings when necessary.

<Composition for labeling lesions for biocompatible purposes>

As a means for achieving the above object, the present invention is a microsphere particle containing a biocompatible polymer and a dye for staining living tissue; and a surface modifier coated on the surface of the microsphere particles, and is directly administered to the target lesion during surgery for the target region to confirm the size and location of the target lesion in real time.

The tissue to be labeled by the composition is preferably a solid tissue such as a solid cancer into which the composition can be penetrated and fixed, more preferably prostate cancer, breast cancer, cervical cancer, skin cancer, cervical cancer, lung cancer, brain tumor, gastrointestinal tumor, liver cancer, It may be soft tissue sarcoma, lymphoma, etc., but is not particularly limited to cancer or other benign lesions containing tissues into which the synthetic material can penetrate and be fixed.

The term "biocompatible polymer" of the present invention is a polymer that is non-toxic or less toxic in the body and can be used for medical purposes, such as "poly(methyl methacrylate) (PMMA)". After the biocompatible polymer of the present invention is injected into the lesion tissue in the body and labeled, it can be used to stay in the lesion for a long time and confirm the size and location of the lesion tissue.

Here, the biocompatible polymer is polymethyl methacrylate (PMMA), polyglycolic acid (PGA), polylactic acid (PLA), polylactic acid-polyglycolic acid copolymer (polylactic acid- polyglycolic acid copolymer (PLGA), poly-ε-caprolactone (PCL), lactic acid-ε-caprolactone copolymer (PLCL), polydioxanone (PDO) ), polytrimethylene carbonate (PTMC), polyamino acid, polyanhydride, polyorthoester, polyphosphazene, polyiminocarbonate, It may be any one of polyphosphoester, polyhydroxyvalate, copolymers thereof, and mixtures thereof. In addition, since the biocompatible polymer embeds a pigment or a fluorescent pigment in the present invention, it may have characteristics of being transparent and transmitting light well.

The term "dye for staining living tissue" of the present invention refers to a substance that binds to a living tissue and marks the bound position, thereby enabling the marked position to be confirmed with the naked eye or using a detection tool. Here, the dye for dyeing the living tissue may be a dye or a fluorescent dye that can be confirmed with the naked eye. For the purpose of the present invention, the pigment for staining biological tissue may be included in a biocompatible polymer in a form combined with a bioprotein and used for labeling a lesion site. Fluorescent dyes that can be generated and detected using equipment such as a fluorescent camera may be used alone or in combination, but are not particularly limited thereto.

As used herein, the term "dye visible to the naked eye" refers to a type of dye that allows a labeled material bound to a biological tissue to display a color in the visible ray region so that a labeled region can be visually confirmed. For the purpose of the present invention, the pigment that can be confirmed with the naked eye can serve to improve the success rate of surgery by injecting the pigment into the lesion site in the body so that the lesion can be visually confirmed in the operating room. The pigments that can be confirmed with the naked eye are preferably neutral, Nile blue, Bismarck brown, lithium carmine, trypan blue, Janus green, methyl violet, auramine, maragaite green, safranin, eosin, congo red, erythrosin, Nigrosin, alcian blue hematoxylin, aniline blue, light green, etc. may be used alone or in combination, but are not particularly limited thereto as long as the target lesion tissue can be identified.

The term "fluorescent dye" of the present invention refers to an organic compound that absorbs light of a certain wavelength to form an excited state, maximizes the light penetration distance, and generates fluorescence capable of minimizing error signals caused by moisture. In other words, it may be a near-infrared fluorescent dye, which is an organic compound that generates fluorescence of a near-infrared wavelength of preferably 700 nm to 3000 nm, more preferably 750 nm to 900 nm. The near-infrared wavelength fluorescence generated from the near-infrared fluorochrome can be photographed or monitored in real time using equipment such as a fluorescence camera, a fluorescence sensing probe (PCT/KR2011/009271), and a surgical fluorescence camera. The fluorescence of the near-infrared wavelength of the present invention has relatively low absorption in the body compared to other wavelength bands, so even near-infrared rays generated in a relatively deep part of the body can be detected outside the body. For the purpose of the present invention, the near-infrared fluorochrome can be injected into the lesion site in the body so that the lesion can be accurately identified using fluorescence imaging equipment in the operating room, thereby improving the success rate of surgery. In particular, unlike the pigments that can be confirmed with the naked eye, since the location of the lesion can be detected outside the body before direct examination of the lesion by incision, it is possible to perform surgery quickly and accurately. Preferably, indocyanine green (ICG) may be used as the near-infrared fluorescent dye, and as long as it is a near-infrared fluorescent dye usable in the human body, it may be included in the scope of the present invention.

The complex in which the near-infrared fluorescent dye is bound to a biocompatible polymer marks the target lesion, and has the advantage of superior stability and accuracy of the detected fluorescence signal compared to the complex in which the near-infrared fluorescent dye is bound to other materials, so that micro-lesions is high, and the accuracy of lesion excision can be improved.

The term "indocyanine green (ICG)" of the present invention refers to a dye for fluorescence imaging in the near-infrared region widely used in biology or medicine. It is advantageous for clinical application as a fluorescent dye that can be used in the human body because it is excreted in feces. In fact, cases of applying indocyanine green to the human body have been reported in several papers, and for example, it has been reported that it was safely used clinically in 18 breast cancer patients (T. Kitai, et al., Breast Cancer, 12:211-215, 2005). Currently, it is already widely used clinically.

Fluorescent pigments such as indocyanine green are ingested from tissues into the liver, decomposed, and excreted through the biliary tract. It is advantageous for pigments such as indocyanine green to be bound to relatively large particles of a certain size (10 to 100 μm) in order not to migrate to veins or lymphatic vessels of surrounding tissues. The microspheres of the present composition can be prepared in a form in which the fluorescent dye is completely encapsulated in the microspheres so that the microspheres stay at the injection site for several months and continue to generate fluorescence. Since the water-soluble fluorescent dye introduced into the microsphere is trapped in the wall of the hydrophobic polymer microsphere, the effect of staying at the injection site for a long time can be expected.

In addition, even if the fluorescent dye is well encapsulated in the microsphere and does not flow to the surroundings, the dye should not lose its properties by being quenched by continuous excitation light or decomposed by a biological reaction. Here, quenching means that when a fluorescent dye is exposed to excitation light, it emits fluorescence in an excited state and falls into a stable state, but is decomposed by the oxygen generated at this time or forms a dimer (diner, tetramer) so that fluorescence does not occur. This means that the fluorescence generating ability is lost. When quenching occurs, even if the dye is present at the injection site, it cannot function as a marker because it does not generate fluorescence. Therefore, in order for the fluorescent dye to be maintained for several months, the fluorescent dye, such as indocyanine green, must be well bound, such as being embedded in microspheres of an appropriate size so as to stay at the injected site, and also have to be less quenched.

Here, the fluorescent dye may be used alone or in combination with a biological protein. That is, in the biocompatible composition for labeling target lesions of the present invention, the microsphere particles may further include a biological protein. When the fluorescent dye forms a conjugation with the biological protein, stronger fluorescence and a longer duration can be exhibited compared to when the fluorescent dye is used alone. Here, the biological protein may be, for example, albumin, but is not limited thereto, and may be included within the scope of the present invention as long as it forms a conjugation bond with the fluorescent dye.

In addition, the composition for labeling the target lesion of the present invention may have a stronger fluorescence intensity as the biological protein is included in a higher concentration. More specifically, the biological protein may be included in a concentration of 0.5 to 20% w/v with respect to the composition for marking the target lesion. Details of the biological protein will be described in more detail below.

The surface modifier may be any one of hyaluronic acid, collagen, and mixtures thereof. Side effects such as foreign body reaction by macrophages or foreign substance giant cells, etc. Since this may occur, in order to reduce the occurrence of such side effects, hyaluronic acid or collagen, which is a biopolymer material widely present in the body, may be used as a surface modifier.

Here, the microsphere particles may have a diameter of 10 to 100 μm. A more preferable diameter may be 30 to 70 μm, and may exhibit an average diameter of 40 μm. Tissues in our body are densely composed of collagen fibers, reticular fibers, and elastic fibers with a thickness of tens to hundreds of nm, and the pore size of the walls of capillaries and lymphatic vessels passing through tissues is also tens of nm. In order for the microsphere particles (ICG-albumin-PMMA microsphere) of the present invention to not flow out of the tissue, a micrometer size larger than the pore size pierced in the wall of the fiber or capillary/lymphatic vessel in the tissue is appropriate, and through animal experiments, the above particle size is confirmed to be effective. In order to obtain the above microsphere particle diameter, the microsphere may be obtained through filtering.

Here, the composition may have an average density of 1 to 3 g/ml. The density of this composition is higher than that of distilled water at room temperature (0.99821 g/ml), and when diluted in distilled water for use as an injection, precipitation may occur. In injections, the occurrence of such precipitates may cause clogging of the needle during injection or make it difficult to uniformly introduce the composition of the present invention into the target lesion. Therefore, the injectable composition containing the composition for marking the target lesion of the present invention may additionally be used by mixing the composition for marking the target lesion with a biocompatible polymer such as hyaluronic acid to suppress the occurrence of precipitation. The average density and the injection composition for the composition for marking the target lesion of the present invention will be described in more detail in the <Injectable composition for marking the target lesion> and {Examples and evaluation} section below.

<Method of manufacturing composition for biocompatible target lesion labeling>

The present invention provides a method for preparing a composition for marking a target lesion as a means for achieving the above object. More specifically, the method for preparing a composition for marking a target lesion of the present invention includes mixing an organic solvent to which a biocompatible polymer is added and distilled water to which a dye for staining living tissue is added; and injecting the mixed solution into a water-soluble solvent to which a surface modifier is added, followed by stirring to form microsphere particles.

The surface modifier may be coated on the surface of the formed microsphere particle.

Here, the organic solvent is any one selected from the group consisting of dichloromethane (DCM), orthoxylene (o-xylene), m-xylene (m-xylene), para-xylene (p-xylene), and combinations thereof. It may, but is not limited thereto.

Here, the water-soluble solvent is polyvinylpyrrolidone (PVP), polyacrylic acid (PAA), polyacryl amide (PAAm), polyhydroxyethyl methacrylate (PHEMA), poly It may be any one selected from the group consisting of polyvinyl alcohol (PVA) and combinations thereof, but is not limited thereto.

Here, the distilled water may be further added with a pigment-biological protein conjugate for staining living tissue.

In the preparation method of the biocompatible target lesion-labeling composition of the present invention, the biocompatible polymer, the pigment for staining biological tissue, the surface modifier, and the biological protein are the same as described above.

1 is a schematic diagram showing a method for preparing a composition for labeling lesions for biocompatible purposes according to the present invention. More specifically, polymethyl methacrylate (PMMA) as the biocompatible polymer, indocyanine green (ICG) as the pigment for staining biological tissue, hyaluronic acid as the surface modifier, and phosphorus serum albumin as the biological protein. (HSA) shows a process of preparing a composition for labeling biocompatible target lesions.

Referring to FIG. 1, it can be seen that the biocompatible composition for labeling lesions for biocompatible purposes prepared according to the above-described manufacturing method appears in a spherical shape with a diameter of 10 to 100 μm, and is in the form of a white powder to the naked eye. there is.

Here, the concentration of the dye for staining living tissue may be 1 to 100 μM. A more preferred concentration may be 3 to 30 μM. In the present invention, as the concentration of the dye for dyeing the living tissue is included in the above-described range, the intensity of fluorescence may appear stronger. Specific details related to the concentration of the dye for dyeing the living tissue will be described in more detail in {Examples and Evaluation} below.

Here, the concentration of the water-soluble solvent may be 0.1 to 10% w/w, and the concentration of the surface modifier may be 0.01 to 0.3% w/v.

In the present invention, in order to coat the microsphere particles with hyaluronic acid or collagen, there may be a method of adding hyaluronic acid or collagen to the raw material of the polymer microspheres, but hyaluronic acid is not heated and melted, and hyaluronic acid is denatured during the heat melting process. It is likely to be. In addition, a method of dissolving the hyaluronic acid in a solvent to form a hyaluronic acid solution and coating the microspheres may be considered, but the hyaluronic acid is not easily soluble in general organic solvents, so it is impossible. In the present invention, a step of preparing a solution in which hyaluronic acid is well dissolved in the step of making microsphere particles was developed to provide a coating of hyaluronic acid. The hyaluronic acid is known to stimulate fibroblasts to produce collagen. The collagen produced in this way can be expected to have an effect that the composition for marking the target lesion injected into the tissue is well adhered to the injection site and does not flow even after several months.

Here, the preparation method of the target lesion-labeling composition may further include selecting microsphere particles having a specific size by washing and filtering the stirring solution. As described above, in order for pigments such as indocyanine green to not move to veins or lymphatic vessels of surrounding tissues, it is preferable to bind to relatively large particles of a certain size (10 to 100 μm). The manufacturing method may further include washing and filtering the stirring solution to select microspheres having a size of 10 to 100 μm.

<Injectable composition for labeling the target bottle>

Furthermore, the present invention is a means for achieving the above object, the composition for labeling the target lesion of claim 1; and hyaluronic acid (HA) having an average molecular weight of 0.5 to 3.0 MDa.

In order to maximize the utilization of the biocompatible polymer containing the pigment for staining biological tissue of the present invention as an in vivo marker, the physical properties of the composition for marking target lesions of the present invention can be enhanced by using the biopolymer. That is, the target lesion marking composition of the present invention serves to clearly recognize the lesion site during surgery by marking the lesion by being injected into the lesion site in the living body. In order to perform efficiently, diffusion must be prevented as much as possible at the site of the injected lesion and a high fluorescence signal must be consistently produced. As one means for achieving this purpose, "hyaluronic acid" can be used. When the composition for target lesion marking of the present invention is mixed with a hyaluronic acid solution, the persistence of the composition in vivo can be improved by the hyaluronic acid solution.

In addition, in the injection composition for target lesion marking of the present invention, when the hyaluronic acid is additionally mixed with the injection, it is possible to prevent injection failure due to clogging of the syringe needle by the precipitated microspheres by slowing down or blocking the precipitation of the microspheres. . Contents related to this will be described in more detail in {Examples and Evaluation} below.

Here, based on 100% by weight of the injectable composition, hyaluronic acid (HA) may be included in an amount of 0.3 to 1% by weight. When the hyaluronic acid is included in an amount of less than 0.3% by weight, the composition for marking the target lesion is precipitated, and the precipitated composition may cause clogging of the syringe needle. On the other hand, when the hyaluronic acid exceeds 1% by weight, excessive viscosity is imparted to the injectable composition, which may also cause discomfort during injection. Accordingly, the hyaluronic acid is preferably included in an amount of 0.3 to 1% by weight based on 100% by weight of the injectable composition of the present invention. Specific details related to this will be described in more detail in {Examples and Evaluation} below.

Here, the hyaluronic acid (HA) may have a viscosity of 24 to 1500 cps at room temperature. When the viscosity of the hyaluronic acid is less than 24 cps, the viscosity of the injectable composition of the present invention is too low, so that it is difficult to control the injection amount during injection or precipitation may occur. On the other hand, when the viscosity exceeds 1500 cps, excessive force applied to the syringe is required due to the too high viscosity, so that injection may not be easy. Accordingly, the viscosity of the hyaluronic acid is preferably 24 to 1500 cps at room temperature.

Here, the target lesion to which the injectable composition is injected may maintain a label for several months or more. More specifically, it may be preferable that the labeling of the target lesion through the injectable composition be maintained for 4 to 12 months. After the composition is injected into the target lesion before pre-cancer treatment (pre-operative chemotherapy), the composition should maintain fluorescence even after the pre-chemo treatment so that follow-up of the target lesion is possible. The specific period of the pre-cancer treatment varies depending on the type of cancer and the type of chemotherapy, and may change according to the development of chemotherapy, but can generally be performed for a period of about 4 to 8 months. Accordingly, it is preferable that the label (fluorescence) of the target lesion injected with the injection composition for marking the target lesion is maintained for 4 months or more, more preferably 6 months or more, and even more preferably 8 months or more. . Details related to the stability of the injectable composition in the target lesion will be described in more detail in {Examples and Evaluation} below.

<Method of providing information on the location of the target lesion>

In addition, the present invention provides a method for providing information on the location of a target lesion using the above-described composition for marking the target lesion. More specifically, the present invention comprises the steps of directly administering the composition for marking the target lesion according to the present invention to the target lesion or around the target lesion; And recognizing the position where the color changes from the tissue to which the composition for marking the target lesion is administered as the target lesion, and confirming in real time during operation of the marked area; a method for providing information on the location of the target lesion, including to provide.

When the composition for marking a target lesion provided in the present invention is administered to a lesion tissue in a living body, the position of the lesion can be marked through color, near-infrared fluorescence, or a combination thereof, and the position and size of the target lesion can be detected by detecting the mark. Since the back can be detected in real time during surgery, it is possible to improve accuracy when removing a lesion for surgical purposes and to prevent excessive loss of normal tissue.

In addition, since the composition for target lesion marking of the present invention contains a biocompatible polymer, it can remain for a long period of time while showing a high fluorescence signal in the injection lesion in vivo compared to a complex in which a dye for dyeing a living tissue is bound to another material. , the accuracy of surgical resection of the target lesion can be easily confirmed. For example, by injecting the composition of the present invention into the location of the microlesion before surgery, it is possible to stably and accurately check the lesion site with the naked eye and a fluorescence camera even after several days to weeks of surgery.

Hereinafter, with reference to the accompanying drawings and embodiments will be described in more detail with respect to what the present specification claims. However, the drawings and embodiments presented in this specification can be modified in various ways by those skilled in the art and have various forms, and the description in the present invention is not to limit the present invention to a specific disclosed form. Rather, it should be regarded as including all equivalents or substitutes included in the spirit and technical scope of the present invention. In addition, the accompanying drawings are presented to help those skilled in the art to more accurately understand the present invention, and may be exaggerated or reduced than actual.

{Example and evaluation}

<Example>

Preparation Example 1. Preparation of a solution in which human serum albumin (HSA) is bound to indocyanine green (ICG)

A solution of 3.2 mM concentration prepared by dissolving 25 mg of indocyanine green (ICG) in 10 ml of distilled water is prepared. 312.5 μl of this 10 ml solution was transferred to a 1.5 ml Eppendorf tube and diluted with 687.5 μl of distilled water to prepare 1 ml of a 0.1 mM solution containing 78 μg of ICG. Meanwhile, when 0.4 ml of a 20% phosphorus serum albumin (HSA) solution is prepared and diluted with 0.6 ml of distilled water, an 8% solution having a volume of 1 ml is prepared. When 125 μl of each of the prepared 0.1 mM ICG 1 ml and 8% HSA 1 ml solutions is transferred to one 1.5 ml Eppendorf tube, 0.25 ml of a solution in which 50 μm ICG and 4% HSA are combined is finally prepared.

Preparation Example 2. Preparation of polymethyl methacrylate (PMMA) solution

It was prepared by completely dissolving 300 mg (15 pieces) of ACRYPET polymethyl methacrylate beads (PMMA bead, Lotte MRC) in 3 ml of dichloromethane (DCM) solution.

Preparation Example 3. Preparation of Polyvinylpyrrolidone (PVP) Suspension Stabilizer Solution Containing Hyaluronic Acid

4 g of polyvinylpyrrolidone (PVP) was completely dissolved in 100 ml of distilled water to prepare a 4% concentration polyvinylpyrrolidone (PVP) solution, and then 0.15 g of hyaluronic acid was added and suspended. A stabilizer solution was prepared.

Preparation Example 4. Production of polymethylmethacrylate (PMMA) microsphere particles combined with indocyanine green (ICG)-phosphorus serum albumin (HSA)

0.25 ml of the ICG-HSA aqueous solution prepared in Preparation Example 1 and 3 ml of the PMMA solution prepared in Preparation Example 2 were put in a 15 ml tube, and the aqueous solution layer and the organic solution layer were mixed at room temperature at a speed of 7500 rpm for 20 minutes using a homogenizer. Mix vigorously with each other. This mixture was added to 40 ml of a suspension stabilizer solution (Preparation Example 3) being stirred at room temperature at 900 rpm. After stirring for 90 minutes to blow off the organic solvent gas, the mixture was divided into 2 ml Eppendorf tubes and washed three times for 10 minutes at 2000 rpm using a centrifuge. Combine all the dispersed ICG-HSA-PMMA microspheres into one tube. Filter this material with 70 ㎛ and 30 ㎛ size filters to obtain a product with a size of 30-60 ㎛. After filtering, powdered ICG-HSA-PMMA dried at room temperature is put into a gamma irradiator and disinfected at 75 Gy for 17 seconds.

The process of the above embodiment is shown schematically in FIG. 2 . Figure 2 is a schematic diagram showing in detail the manufacturing method of the composition for labeling biocompatible lesions of the present invention. Referring to the above-described Examples and FIG. 2, the biocompatible composition for labeling lesions for the purpose of the present invention was prepared.

<evaluation>

1. Evaluation of physical properties of ICG-HSA-PMMA

1-1. Property evaluation

Figure 3 (a) is a real picture of PMMA including ICG and HSA and a picture taken with a fluorescence camera, and Figure 3 (b) is a picture taken with a fluorescence microscope of PMMA according to the presence or absence of ICG and HSA. Referring to Figures 3 (a) and 3 (b), it can be confirmed that the composition for labeling the target lesion of the present invention exhibits fluorescence, and as the composition includes a biocompatible polymer (PMMA), spherical microspheres It can be confirmed that the particle is represented.

1-2. density evaluation

Since it is difficult to directly measure the density in an actual use form in an aqueous solution state, the density of the microspheres was measured indirectly by referring to the change in density according to the concentration. Density was measured using microspheres of representative ICG 6.5 μM 4% HSA prepared above, and using microspheres with a concentration of about 20 mg / ml to 200 mg / ml (2 to 20% (w / v)), the following Density was measured indirectly as

More specifically, at room temperature, after filling a transparent container with 20 ml of distilled water, the above-described microspheres were administered, and the density of the aqueous solution containing the microspheres was measured. In particular, the density changed according to the concentration of the microspheres measured. For reference, after administration of microspheres in distilled water, it was confirmed that they did not dissolve easily in distilled water and when mixed with shaking, they showed a white suspension. .

Table 1 is a table showing the density of distilled water containing microspheres, and the density according to the concentration of the microspheres is described.

Microspheres % (w/v) Density (g/ml) 25.23 1.0621 18.66 1.0469 12.61 1.0309 10.69 1.0251 9.525 1.0231 5.35 1.0112 2.16 1.0021

A linear regression equation was obtained by linear regression analysis with reference to Table 1, and the density at 100% was measured by extrapolation.

[ceremony]

Figure 4 is a graph showing the density according to the microsphere particle concentration of the present invention with respect to distilled water. Referring to FIG. 4 , the density of the microspheres was estimated to be 1.2574 g/ml according to the above formula. In addition, when the distilled water in which the microspheres were dissolved in distilled water was checked after 20 minutes, it was confirmed that a white suspension had settled in the lower part of the container. As a result, it was determined that the microspheres had a density of 1.2574 g/ml, which was higher than that of distilled water at room temperature (0.99821 g/ml).

1-3. Fluorescence evaluation

In order to confirm the optimal ICG concentration and albumin concentration of ICG-Albumin-PMMA microsphere fluorescent markers showing near-infrared fluorescence, the near-infrared fluorescence of microspheres prepared with various ICG and albumin concentrations was measured.

In order to determine the mixing ratio of ICG and albumin that can exhibit the strongest near-infrared fluorescence, 1.3 to 1032 μM of ICG and 0 to 1% w / v (10 mg / ml) of albumin were reacted in various ratios, and each ICG-albumin -PMMA microspheres were prepared, and the signal strength of near-infrared fluorescence appearing in the microspheres was measured and shown in Table 2.

Albumin (% w/v) ICG µM 0 0.05 0.25 0.5 0.7 One 10 20 1.3 6 14 79 99 140 177 467 577 3.9 40 18 123 153 197 293 730 953 6.5 71 13 148 170 240 327 767 974 9 104 11 152 154 224 284 574 734 12.9 137 9 121 125 143 193 330 484 25.8 153 4 85 90 113 150 200 317 38.7 142 5 54 60 77 107 133 157 51.6 133 3 34 42 50 83 97 117 64.5 125 5 25 32 40 60 64 84 77.4 111 5 18 25 33 37 37 50 103 80 8 13 20 27 31 33 37 258 40 10 5 12 20 20 22 22 516 24 4 One 8 15 16 18 20 774 13 2 One 7 11 14 17 16 1032 10 2 One 6 7 8 9 8

The results of Table 2 are shown in FIG. 5 as a graph. Figure 5 is a graph showing the change in near-infrared fluorescence signal intensity of microspheres according to the concentration change of ICG and albumin.

Referring to Table 2 and FIG. 5, when albumin was not added, the intensity of near-infrared fluorescence of microspheres could be confirmed that ICG of 25.8 μM represents the highest value of near-infrared fluorescence signal intensity, and 0.05% w / v (0.5 In the case of treatment with albumin of mg/ml), 3.9 μM of ICG showed the highest value of NIR fluorescence signal intensity, but it can be seen that overall fluorescence was remarkably low. When 0.25% w/w (2.5 mg/ml) of albumin was treated, 9 μM of ICG showed the highest NIR fluorescence signal intensity, and when the concentration of albumin was higher, 0.5% w/v (5 mg/mL), 0.7% w/v (7 mg/mL), 1% w/v (10 mg/mL), 10% w/v (100 mg/mL), 20% w/v (200 mg/mL) In the case of treatment with albumin of ㎖), 6.5 μM of ICG may exhibit the highest value of near-infrared fluorescence signal intensity.

Judging from the above experimental results, the concentration of ICG having the strongest intensity of fluorescence at the same albumin concentration was 6.5 to 25.8 μM. Therefore, in the present invention, the ICG concentration range is preferably 1 to 100 μM, and more preferably 3 to 30 μM.

In addition, at the same ICG concentration, the intensity of fluorescence continued to increase up to 20% w/v, the highest albumin concentration used in the experiment. Realistically, since the maximum concentration of human serum albumin required for the production of microspheres was 20% w/v, microspheres could not be prepared with a higher albumin concentration. Therefore, the experimental results did not confirm the concentration of albumin at which the intensity of fluorescence is maximized, but from the experimental results, it can be confirmed that the maximum fluorescence starts to increase from microspheres with 0.5% albumin than microspheres without albumin. , Therefore, albumin used for microsphere preparation is ideally 0.5% w/v or more, and may be suitable for microsphere preparation up to 20% w/v, the highest experimentally possible albumin concentration.

2. Evaluation of usefulness of the injection composition of ICG-HSA-PMMA

2-1. Evaluation of usefulness of hyaluronic acid

In order to determine the usefulness of hyaluronic acid as an injectable composition, the suspension retention time of the ICG-Albumin-PMMA/hyaluronic acid composition according to the concentration of hyaluronic acid was measured. ICG-Albumin-PMMA was used at a concentration of 2 mg/ml, and the final injection of ICG-Albumin-PMMA microspheres showed the appearance of a very light green or white suspension when completely dissolved in the hyaluronic acid composition.

More specifically, the time for maintaining the suspension after the ICG-Albumin-PMMA injection composition was prepared was measured, and the maintenance of the suspension according to the concentration of the added hyaluronic acid was measured. Here, the suspension retention time was defined as the time point at which the criterion for distinguishing the sinking and floating areas based on the transmittance of 500 nm light of 20% is visible. And, the results are described in Table 2.

Table 3 is a table showing the time for which the suspension property is maintained after the preparation of the ICG-Albumin-PMMA microsphere injection according to the concentration of the hyaluronic acid solvent.

Concentration of hyaluronic acid solvent (%, w/v) Suspension holding time 0 (simple injection water, distilled water) within 10 minutes 0.1 within 60 minutes 0.2 within 3 hours 0.3 within 4 hours 0.4 within 12 hours 0.5 within 24 hours 1.0 more than 24 hours 1.5 more than 24 hours 2.0 more than 24 hours 2.5 more than 24 hours

Referring to Table 3, it can be seen that the injectable composition without hyaluronic acid had a relatively shorter suspension retention time than the hyaluronic acid-added composition, and the suspension retention time increased as the proportion of hyaluronic acid added increased. there was.

On the other hand, when 0.1% of hyaluronic acid was added to the injectable composition, the suspension retention time increased to less than 60 minutes, but the suspension was 1 to 3 hours from the hyaluronic acid concentration of 0.2% (w / v), and the hyaluronic acid From a concentration of 0.3% (w/v), it was confirmed that the suspension lasted for more than 3 hours. In the case of actual clinical injection, since the time to prepare the injection and actually inject it is less than 1 hour, it was judged to be effective as an actual injection formulation from 0.2% (w / v).

6 is a photograph showing the degree of dispersion of microspheres according to the concentration of hyaluronic acid in the injectable composition for labeling biocompatible lesions of the present invention. More specifically, the pictures were taken immediately after preparing the ICG-HSA-PMMA/hyaluronic acid injection prepared above and after 30, 60, and 120 minutes. Left a) is an injection in which water for injection (distilled water) is mixed with microspheres. Right b) is a photograph of the microsphere injection prepared with 0.3% hyaluronic acid.

Referring to FIG. 6, it can be seen that the injectable composition prepared in water for injection quickly sinks, whereas the injectable composition to which 0.3% hyaluronic acid is added does not completely sink for a long time (up to 120 minutes). That is, from the results of FIG. 3, it means that the ICG-HSA-PMMA/hyaluronic acid injectable composition in which 0.3% of hyaluronic acid is added to microspheres is very useful as an injectable composition because it will not sink quickly during preparation for injection.

2-2. Evaluation of usefulness as an injectable composition

Depending on the concentration of hyaluronic acid, the force applied to the finger during injection (gliding force, N) was measured under various conditions, and the usefulness of ICG-Albumin_PMMA/hyaluronic acid as an injection composition was judged.

More specifically, when the finally manufactured injection is used as an injection, the ease of injection can be improved by using 21G, 22G, 23G, 26G needles (Kovax, Korea Vaccine Co., Ltd.), 20G spinal needle (Taechang Industrial Co., Ltd.) and 7Fr endoscope. It was evaluated using a 3cc disposable syringe (3cc, BD, Luer-Lock Tip, Singapore) equipped with each syringe (20G (needle inner diameter: 0.603mm), 21G (needle inner diameter: 0.495mm), 22G (needle inner diameter: 0.394 mm), 23G (needle inner diameter: 0.318mm), 26G (needle inner diameter: 0.241mm))

The force applied to the syringe handle for each injection needle (force applied to the finger during injection, N) was measured. In addition, resistance was measured using an endoscopic syringe (7 Fr, length 180 cm, MTW, Germany) having a 1.8 m connecting tube.

The injection used in the experiment used a concentration of 2 mg/ml of ICG-Albumin-PMMA microspheres, and the concentration of hyaluronic acid in the final injection was set at 0.1 to 3.0% (w/v). After injecting the injection into the syringe, the experiment was immediately conducted so that the microspheres did not sink, and while the final injection was injected at a rate of 0.1 cc/sec, the strain was measured using a strain-gauge dynamometer (Digital Force Gauge DPU-500N , IMADA, Toyohashi, Japan). And, the results are described in Table 4 below.

N Blank
syringe Distilled water Hyaluronic acid (% (w/v)) [ICG-Albumin-PMMA microspheres 2mg/ml] 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.5 2.0 2.5 3 26G 1.2 2.9 3.0 3.3 3.5 4.3 4.9 5.2 5.9 6.1 6.5 6.6 7.9 9.9 12.6 15.8 23G 1.9 2.3 2.4 2.5 2.7 3.0 3.5 3.8 4.1 4.2 4.4 4.6 6.5 8.6 10.2 12.8 22G 1.6 1.8 1.8 2.2 2.6 2.7 3.1 3.3 3.6 3.9 3.8 4.0 6.6 8.3 9.1 11.8 21G 1.8 1.9 1.9 2.2 2.5 2.7 2.7 2.9 3.1 3.2 3.4 3.5 4.6 6.6 7.9 9.5 20G 1.8 1.7 1.8 2.5 3.1 3.4 3.5 3.8 3.9 4.5 4.7 4.9 8.4 12.8 17.6 19.6 7Fr 1.9 3.3 3.4 7.5 12.1 17.8 21.6 25.4 27.2 33.4 36.9 39.5 not measurable not measurable not measurable not measurable

In general, when performing an injection in a clinical setting, a force of 0.6 to 1.4 N was applied to the finger at the time of injection. The average adult's grip strength is 400 N, and the grip strength that can be handled by fingers is weaker. A force gauge was installed between the thumb and index/middle finger in 10 men and women, and the maximum force (MaxPower) and the comfortable injection force (EasyPower) were measured when the maximum force was applied. The maximum power (MaxPower) was 98.1 N (113.5 ~ 78.2, standard deviation 9.8) in adult males and 43.1 N (50.6 ~ 34.2, standard deviation 6.1) in adult females, and 25.4 N (29.7 ~ 20.1 standard deviations 3.3 standard deviations) in adult males when applying force comfortably. ), and adult females were 14.5 N (18.3 to 11.5 standard deviation 2.2). The resistance of the ideal injection was judged based on the maximum pressure of 43 N when a female adult injected and 14.5 N when giving force comfortably. That is, the maximum injection force was determined to be 40 N, and the resistance to comfortably and ideally be injected was determined to be 10 N.

In the case of injection using a general injection needle (21 ~ 26G), there was no difficulty in use because the resistance during injection was 10N up to 1% concentration of hyaluronic acid. In the case of a 20G needle thickness and much longer than other needles, the puncture needle did not exceed 10N in 1%.

On the other hand, in the case of using an endoscopic syringe needle (7Fr, 180cm), which requires a lot of resistance due to the long length of the conduit, the force for injection was 1.9 N, and when the hyaluronic acid concentration was 1% (w/v), the force was 39.5 The injection was possible only when a significantly higher pressure was applied due to the force of N, and it was impossible to control the injection amount or speed.

Although there are individual differences, it is difficult to inject precisely when a force of 40 N is applied to the finger because it is difficult to inject into the tissue. On the other hand, when an endoscope syringe needle (7Fr, 180 cm) was used, when the hyaluronic acid content exceeded 1%, the resistance increased (> 40 N), making it impossible to inject with bare hands.

Under the condition of hand injection with maximum force (resistance of 40 N or less), it was possible to inject up to 3% (w/v) or more of hyaluronic acid, and in the case of a puncture needle (20G) with a long needle, 3% (w/v) until was possible. Even in the case of an extremely long special syringe (endoscopic syringe) connected by a conduit from the syringe to the needle, it was possible to inject up to 1.0% (w/v) without difficulty.

On the other hand, in the case of using a general syringe (26G to 20G) that directly contacts the tissue, it is preferable to gently inject into the tissue, and ideally a force of less than 10 N is applied. In the case of hyaluronic acid, it was possible to inject up to 2% (w/v) with a general needle (21 ~ 26G) with a force of less than 10 N, and in the case of a puncture needle (20G), it was possible to inject up to 1.5% (w/v). The endoscopic syringe was able to inject up to 0.2 to 0.3% (w/v) without difficulty.

2-3. Assessment of syringe pressure change with sedimentation

The pressure (A) when the injection composition to which hyaluronic acid is added is immediately injected, and the pressure (B) applied to the syringe at the start of the injection after the composition precipitates after the injection composition is left for 120 minutes, and the The pressure (C) was measured and described in the table below.

At this time, the injection used in the experiment used a concentration of 2 mg / ml of ICG-Albumin-PMMA microspheres, and the concentration of hyaluronic acid in the final injection was set at 0.1 to 3.0% (w / v). A 26G syringe was used and the unit of pressure applied to the syringe was measured in N. In addition, the unit of sliding force was measured in N (Digital Force Gauge DPU-500N, IMADA, Toyohashi, Japan).

26G Distilled water Hyaluronic Acid (% (w/v)) 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.5 2.0 2.5 3 A 2.9 2.9 3.1 3.5 4.2 4.7 5.3 5.8 6.2 6.3 6.7 7.7 9.7 12.4 15.6 B ND 45.2 34.7 3.7 4.3 4.8 5.4 5.7 6.3 6.5 6.9 7.5 9.8 12.2 15.8 C ND 3.5 3.6 3.5 4.2 4.3 5.2 5.3 6.0 6.2 6.5 7.3 9.5 12.5 15.8 B-A ND 42.3 33.6 0.2 0.1 0.1 0.1 -0.1 0.1 0.2 0.2 -0.2 0.1 -0.2 0.2

Referring to Table 5, the pressure (A) when the injectable composition is directly injected and the pressure (B) applied to the syringe after the composition is precipitated after leaving the injectable composition for 120 minutes are as the concentration of hyaluronic acid increases, the pressure It was confirmed that (A, B) increased. There was almost no difference between A and B after 0.3% (-0.2 ~ 0.2 N). As the concentration of hyaluronic acid is higher than 0.3%, the injection composition is left for 120 minutes, and then the pressure applied to the syringe (B) and the injection composition are immediately injected. There was little difference from the pressure (A) in the case of The average B-A value was 0.07N from when the concentration of hyaluronic acid was 0.3% (w/v) to 3.0% (w/v), and the standard deviation thereof was 0.15 N.

Looking at the data of 0.1% and 0.2% of hyaluronic acid in Table 5, after leaving for 2 hours, the pressure is 45.2 and 34.7 N, which means that the injection needle is blocked by the injection. After the injection was moved, the pressure rapidly decreased, and there was no difference from the pressure (A) when the injection was immediately injected.

That is, by adding an appropriate amount (0.3% or more) of hyaluronic acid, the precipitation rate could be slowed down, and the injection could be started by applying excessive force because the needle was blocked by the precipitated composition.

On the other hand, when the concentration of hyaluronic acid exceeds 1% (w/v), it can be confirmed that the pressure during injection increases due to the viscosity of hyaluronic acid. It may cause inconvenience in use as an injection.

3. Evaluation of stability of ICG-HSA-PMMA

Since PMMA microsphere particles combined with ICG and HSA capable of generating near-infrared fluorescence signals were expected to serve as markers that act more stably in vivo, the microsphere particles were prepared and tested under both in vitro and in vivo conditions. stability was confirmed.

3-1. Confirmation of in vitro near-infrared fluorescence signal stability

7 is a graph showing a comparison of changes in the intensity of near-infrared fluorescence signals of ICG, ICG-HSA, and ICG-HSA-PMMA particles over time under in vitro conditions measured by IVIS (Perkin Elmer, Hopkinton, MA).

Referring to FIG. 7, according to an embodiment of the present invention, when preparing PMMA microspheres (ICGHSA-PMMA) combined with ICG-HSA, a mixing ratio suitable for preparing microsphere particles exhibiting a high level of near-infrared fluorescence signal is 50 μM. It was confirmed that ICG was combined with 4% HSA and mixed with 300 mg of PMMA beads. When bound to biopolymers such as albumin in this way, ICG alone shows stronger fluorescence and duration than when embedded in microspheres.

8 shows the results of measuring the intensity of the near-infrared fluorescence signal emitted from the ICG-HSA-PMMA particles prepared in Preparation Example 4 under in vitro conditions for 5 months by IVIS. More specifically, FIG. 8 (a) is a photograph showing the comparison of stability of ICG-HSA-PMMA particles in HSA and distilled water over time. 8 (b) is a photograph showing the stability of ICG-HSA-PMMA particles over time after putting them in an agarose gel.

Referring to FIG. 8 (a), the PMMA-containing particles showed stability with a relatively small decrease in the intensity of the near-infrared fluorescence signal. In addition, referring to FIG. 8 (b), the manufactured particles were placed in (a) 20% HSA, distilled water, and (b) agarose gel, and the near-infrared fluorescence signal emitted for 17 weeks was measured with a fluorescence camera to show stability with the naked eye. confirmed that

3-2. Confirmation of in vivo near-infrared fluorescence signal stability

The ICG-HSA-PMMA microsphere particles prepared in Preparation Example 4 were put in a hyaluronic acid solution or distilled water, and 100 μl each was injected into the back of an ICR mouse, and the intensity of the near-infrared fluorescence signal generated in each mouse for 4 months was measured using IVIS and fluorescence camera equipment.

9 is a result of IVIS measurement and comparison of the intensity change of near-infrared fluorescence signal over time when ICG-HSA-PMMA particles injected into ICR mice were injected with hyaluronic acid or distilled water in vivo.

Referring to FIG. 9, as a result of IVIS observation, the ICG-HSA-PMMA/hyaluronic acid mixture showed a high-intensity near-infrared fluorescence signal and maintained stability, but the mixture mixed with distilled water showed a very low fluorescence signal.

10 shows the change in the intensity of the near-infrared fluorescence signal over time when the ICG-HSA-PMMA particles injected into the ICR mouse were injected with hyaluronic acid or distilled water under in vivo conditions, photographed with a fluorescence camera and analyzed with the Image J program. This is the result of taking a graph and a mouse with a fluorescence camera.

Referring to FIG. 10, in the case of a fluorescence camera, the fluorescence intensity was measured by measuring with the Image J program after shooting, and both mixtures showed a high intensity near-infrared fluorescence signal, but ICG-HSA-PMMA/hyaluronic acid The stability of the mixture was maintained, while the distilled water mixture showed a gradually decreasing fluorescence signal.

In addition, referring to FIGS. 9 and 10 , when the ICG-HSA-PMMA microsphere particles are mixed with the hyaluronic acid solution, it can be confirmed that the tissue persistence of the microsphere particles in vivo is improved by the solution. The hyaluronic acid serves to collect the complexes injected into the living body and increase the fluorescence signal, thereby maximally preventing the complexes injected into the living body from spreading and obtaining a high fluorescence signal for several months. Therefore, the injectable composition of the complex of the present invention may further include hyaluronic acid.

The composition for marking the target lesion of the present invention, when injected into the target lesion, stays continuously for up to several months and can detect the location and size of the target lesion in real time during surgery, thereby improving the success rate of surgical operation of the target lesion as well as However, since it can prevent excessive loss of normal tissue, it can be widely used as an effective surgical marker. In the experiment, only the shooting results of up to 20 weeks were disclosed, but this was due to other circumstances such as the termination of the experiment according to the experimental regulations related to animal welfare. In addition, although not shown in the graph, it was confirmed that the fluorescence of microspheres lasted for more than one year in vitro.

The above description is merely an example of the technical idea of the present invention, and various modifications and variations can be made to those skilled in the art without departing from the essential characteristics of the present invention.

Therefore, the embodiments disclosed in the present invention are not intended to limit the technical idea of the present invention, but to explain, and the scope of the technical idea of the present invention is not limited by these embodiments. The protection scope of the present invention should be construed according to the claims below, and all technical ideas within the equivalent range should be construed as being included in the scope of the present invention.

Claims (24)

microspheres containing biocompatible polymers and pigments for staining biological tissues; and
Including; surface modifier coated on the surface of the microsphere particles,
A composition for marking a target lesion, which is directly administered to a target lesion during a marked site operation to confirm the size and location of the target lesion in real time. According to claim 1,
The biocompatible polymer is polymethyl methacrylate (PMMA), polyglycolic acid (PGA), polylactic acid (PLA), polylactic acid-polyglycolic acid copolymer (polylactic acid-polyglycolic acid) acid copolymer (PLGA), poly-ε-caprolactone (PCL), lactic acid-ε-caprolactone copolymer (PLCL), polydioxanone (PDO) , polytrimethylene carbonate (PTMC), polyamino acid, polyanhydride, polyorthoester, polyphosphazene, polyiminocarbonate, poly Phosphor ester (polyphosphoester), polyhydroxyvalate (polyhydroxyvalate), copolymers thereof, and any one of mixtures thereof, characterized in that, the composition for target lesion marking. According to claim 1,
The pigment for staining the biological tissue is characterized in that the pigment or fluorescent pigment that can be confirmed with the naked eye, the composition for marking the target lesion. According to claim 3,
The pigments that can be confirmed with the naked eye are neutral, Nile blue, Bismarck brown, lithium carmine, trypan blue, Janus green, methyl violet, o-lamin, maragaite green, safranin, eosin, congo red, erythrosin, nigrosine, Characterized in that any one selected from the group consisting of alcian blue hematoxylin, aniline blue, light green and combinations thereof, the composition for marking the target lesion. According to claim 3,
Characterized in that the fluorescent dye is a near-infrared fluorescent dye, the composition for labeling the target lesion. According to claim 3,
Characterized in that the fluorescent dye is indocyanine green (ICG), the composition for labeling the target lesion. According to claim 3,
The composition for labeling the target lesion, characterized in that the fluorescent dye is used alone or conjugated with a biological protein. According to claim 1,
The surface modifier is characterized in that any one of hyaluronic acid (hyaluronic acid), collagen (collagen) and mixtures thereof, the target lesion marking composition. According to claim 1,
The microsphere particle further comprises a biological protein, the composition for labeling the target lesion. According to claim 9,
The biological protein is characterized in that the albumin (albumin), the target lesion labeling composition. According to claim 1,
The microsphere particles are characterized in that having a diameter of 10 to 100 ㎛, the composition for target lesion marking. According to claim 1,
The composition is characterized in that the average density is 1 to 3 g / μl, the composition for marking the target lesion. mixing an organic solvent to which a biocompatible polymer is added and distilled water to which a dye for staining living tissue is added; and
Forming microsphere particles by adding the mixed solution to a water-soluble solvent to which a surface modifier is added and then stirring,
A method for producing a composition for marking a target lesion, characterized in that the surface modifier is coated on the surface of the microsphere particle. According to claim 13,
Method for producing a composition for labeling a target lesion, further comprising: washing and filtering the stirring solution to select microsphere particles of a specific size. According to claim 13,
The distilled water is a method for producing a composition for marking a target lesion, characterized in that a pigment for biological tissue staining-biological protein conjugate is further added. According to claim 13,
The organic solvent is any one selected from the group consisting of dichloromethane (DCM), orthoxylene (o-xylene), m-xylene (m-xylene), para-xylene (p-xylene), and combinations thereof Characterized in, a method for producing a composition for labeling a target lesion. According to claim 13,
The method for producing a composition for marking a target lesion, characterized in that the concentration of the dye for staining the living tissue is 1 to 100 μM. According to claim 13,
The water-soluble solvent is polyvinylpyrrolidonem (PVP), polyacrylic acid (PAA), polyacryl amide (PAAm), polyhydroxyethyl methacrylate (PHEMA), polyvinyl Characterized in that any one selected from the group consisting of alcohol (polyvinyl alcohol, PVA) and combinations thereof, a method for producing a composition for marking a target lesion. According to claim 13,
The concentration of the water-soluble solvent is 0.1 to 10% w / w, and the concentration of the surface modifier is 0.01 to 0.3% w / v, characterized in that, the method for producing a composition for target lesion marking. The composition for labeling the target lesion of claim 1; and
An injectable composition for labeling a target lesion, comprising hyaluronic acid (HA) having an average molecular weight of 0.5 to 3.0 MDa. According to claim 20,
Based on 100% by weight of the injectable composition, hyaluronic acid (HA) is contained in 0.3 to 1% by weight, the injectable composition for target lesion labeling. According to claim 20,
The hyaluronic acid (Hyaluronic acid; HA) has a viscosity of 24 to 1500 cps at room temperature, the injectable composition for marking the target lesion. According to claim 20,
The injectable composition for marking the target lesion, characterized in that the target lesion injected with the injectable composition is maintained for 4 to 12 months. Directly administering the composition for marking the target lesion of claim 1 to the target lesion or around the target lesion; and
A method for providing information on the location of a target lesion, comprising: recognizing the position where the color changes from the tissue to which the composition for marking the target lesion is administered as the target lesion and confirming it in real time during surgery at the marked site.